Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Summary

An efficient procedure to assess the oligomerization propensity of single-pass transmembrane domains (TMDs) is described. Chimeric proteins consisting of the TMD fused to ToxR are expressed in an E. coli reporter strain. TMD-induced oligomerization causes dimerization of ToxR, activation of transcription and production of the reporter protein, -galactosidase.

Abstract

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.^1-3 One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.^4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.^6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,⁸ direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.⁹ The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).

In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.^10-13 The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where β-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for β-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).

Protocol

1. Cloning Considerations

1. Commercially prepared oligonucleotides representing the TMD of interest flanked by NheI and BamHI restriction sites and 5'-phosphorylated can be ligated into pTox7 (modified in our laboratory by insertion of one base pair directly after the BamHI restriction site¹⁴) (Figure 3) digested sequentially with BamHI and NheI. An example oligonucleotide is shown below:
   5'ctagcTMDSEQUENCEg3'
   3' gTMDSEQUENCEcctag5'

The TMD sequence should be 12-24 residues (shorter sequences will presumably be elongated by vector encoded hydrophobic residues). In order to investigate the int....

Discussion

The ToxR transcriptional reporter assay is a facile way to identify transmembrane sequences with the potential to oligomerize. Since the interactions are occurring within the bacterial inner membrane, this assay circumvents the issues associated with the validity of studying systems in membrane-mimetic environments. Given that cloning of multiple TMDs into a single plasmid can readily be done in parallel and the entire assay can be carried out in 96-well plate format, this assay can be used for high throughput analysis .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BamHI restriction enzyme	Invitrogen	15201023	Invitrogen enzymes were found to be more efficient than alternative suppliers
NheI restriction enzyme	Invitrogen	15444011	Invitrogen enzymes were found to be more efficient than alternative suppliers
15 mL culture tubes	Fisher Scientific	14-956-1J
SOC media	TEKnova, Inc.	S0225	Made up to the appropriate volume and sterilized by autoclaving.
LB media	Sigma-Aldrich	L7275	Made up to the appropriate volume and sterilized by autoclaving.
Chloramphenicol	Sigma-Aldrich	CO378	Stock solution of 30 mg/ mL in ethanol stored in freezer
Arabinose	Fluka	10839	Stock solution of 2.5% (w/v) in water stored in freezer
Na2HPO4	Sigma-Aldrich	S9390
NaH2PO4	Sigma-Aldrich	S9638
KCl	Mallinckrodt Baker Inc.	6858-06
MgSO4.7H2O	Sigma-Aldrich	63138
Sodium dodecylsulfate (SDS)	Sigma-Aldrich	L6026
2-Nitrophenyl β-D-galactopyranoside (ONPG)	Sigma-Aldrich	73660
Z-buffer			16.1 g Na2HPO4 5.5g NaH2PO4 0.75g KCl 0.246g MgSO4 Make up to 1 l, pH 7.0
Z-buffer/chloroform			200 mL β-mercapt–thanol, 2 mL chloroform, make up to 20 mL with Z-buffer. Vortex for 1 min, centrifuge for 1 min at 800 rpm. Make fresh for each plate.
Z-buffer/SDS			160 mg SDS dissolved in 10 mL Z-buffer
Z-buffer/ONPG			40 mg ONPG in 10 mL Z-buffer. Make fresh for each plate
β-mercapt–thanol	Calbiochem	444203
Anti-MBP monoclonal antibody (HRP conjugated)	New England Biolabs	E8038S
Minimal media with maltose			1 x M9 salts, 0.4% maltose, 1 mg/ mL thiamin, 2 mM MgSO4
96-well flat bottom plate	Sarstedt Ltd	83.1835.300
Plate-reader	Beckman Coulter Inc.	DTX880 Multimode Detector
Water bath	VWR international	89032-204
Shaking incubator	Forma Scientific